Cu,Zn superoxide dismutase is a key enzyme in the cellular defense against free radical oxidation. We are carrying out coupled structural and biochemical experiments to probe the contributions of electrostatics, stability, and metal binding to the catalytic mechanism of this enzyme. We have produced mutant proteins with altered electrostatics which either increase or decrease catalytic activity. We have generated other mutants with altered stability. We have also made mutant enzymes with altered metal binding. We will determine the structures of these mutant enzymes to form a basis for interpreting our functional data. These combined results will be important for elucidating the molecular mechanism of neurodegenerative disease (ALS) which has been linked to defects in Cu,Zn superoxide dismutase.